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Specific interactions between proteins regulate nearly all cellular processes. In the nervous system specialized processes such as neuronal proliferation, differentiation and targeting, synapse formation and neurotransmitter release are all tightly controlled by cascades of protein-protein interactions. The extent and nature of these interactions is therefore a question of fundamental importance. The two-hybrid system, which is beginning to be widely applied in many other areas of cell biology, offers a novel and sensitive technique for the identification and analysis of these protein-protein interactions.

J. Neurochem. (2011) 118, 958-967. Abstract Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M1-mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M1-mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-3H]-benzilate ([3H]-QNB) whereas the hydrophilic probe, 1-[ N-methyl-3H] scopolamine ([3H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M1-mAChR antibody also detected both cell-surface and intracellular M1-mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca2+ mobilization were completely inhibited by a cell-impermeable M1 antagonist, muscarinic toxin -7 and the Gq/11 inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of Gq/11 (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M1-mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes. [ABSTRACT FROM AUTHOR]

Purpose: Although α1L-adrenoceptor is recognized as a target of α1 antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine α1L-AR vs α1A-AR tissue distribution because α1L-AR is now considered another phenotype sharing the α1A-AR gene and protein molecule. We labeled the α1A and α1L-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor® 488 (Alexa-488-silodosin) to visualize α1L-AR expression. Materials and Methods: Radioligand binding and functional bioassay experiments were done to assess α1-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed. Results: Although Alexa-488-silodosin had about 10 times lower affinity for all α1-AR subtypes than silodosin in binding and functional studies, it had high selectivity to α1A and α1L-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing α1A-AR but not α1B-and α1D-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin. Conclusions: Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to α1A and α1L-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of α1L-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile. [Copyright &y& Elsevier]

α1-Adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several α1 antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical α1-adrenoceptor subtypes (α1A, α1B, and α1D) have been identified, among which the α1A and α1D-adrenoceptor subtypes have been regarded as the main targets of α1 antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize α1-adrenoceptors pharmacologically, (i.e. all classical α1-adrenoceptor subtypes show high-affinity for the drug). However, we found that α1-adrenoceptors in the LUT show atypical low-affinity for prazosin. Therefore, the concept α1L-receptor, which indicates α1-adrenoceptor(s) showing low-affinity for prazosin has been introduced. A recent study demonstrated that the α1L-adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the α1L-adrenoceptor in the LUT is now re-defined as α1A(L)-adrenoceptor. The physiological and pharmacological difference between classical α1A(H), and α1A(L) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological α1A-adrenoceptor blocking pharmacotherapy. Here, we briefly review the α1-adrenoceptors in the LUT with special reference to phenotype-based (pharmacome) analysis. [ABSTRACT FROM AUTHOR]

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABAB receptor subunit GABAB1. Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABAB2 subunit, their co-expression in HEK293 cells increases levels of GABAB2. GISP also inhibits TSG101-dependent GABAB2 down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABAB2 degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability. [ABSTRACT FROM AUTHOR]

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABAB receptor (GABABR) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABAB1 subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABAB1 subunit via a coiled-coil domain. GISP co-fractionates with GABABR and with the postsynaptic density and co-immunoprecipitates with GABAB1 and GABAB2 from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABABRs. When co-expressed with GABABRs in human embryonic kidney cells, GISP promotes GABABR surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABABRs. [ABSTRACT FROM AUTHOR]